The present invention relates to a filter apparatus for separating objective micro-tissues of an organism such as cells from liquid which contain the micro-tissues, and to a method of separating micro-tissues of the organism using the same apparatus.
There has been performed separation or concentration of the objective cells such as lymphocyte (hereinafter called as "objective cells") from other components, when cells are handled outside of the organism, for example, in the case of culture and preservation of cells as well as in the case of therapy using cells, such as transplanting of bone marrow and hematopoietic stem cells, adoptive immuno-therapy, gene therapy and the like.
In the cell culture and the therapeutic treatment using cells, it has come out as an extremely significant subject to remove cells other than the objective cells, as well as unnecessary liquid components, wastes of cells and products produced by cells. In the case of using the preserved cells, it has also become a significant subject to remove the substance harmful to the cells and the living organism, such as the cryoprotective agent which is used in the cryopreservation. For this reason, there have been proposed various method for cell separation and suitable recovery methods corresponding to the cell separation.
Cell separation methods currently available are roughly classified as follows: (1) precipitation method, centrifugation method, and density-gradient centrifugation method, each using the difference of specific density of cells, (2) electrical separation method using the static charge on the cell-surface, (3) affinity separation method using the specificity of antibody to the antigen existing on the cell surface, and (4) filtration method using the difference in the size and the deformability of the cells, and the like.
However, the methods (1) to (4) respectively have the following problems.
In the precipitation method (1) using the difference in density of the cells, an excessively long time is required to separate the objective cells because the objective cells are separated by using gravity. Therefore, the separation efficiency is unsatisfactory and also the purity or yield is too low.
The centrifugal separation is a method capable of improving the separation efficiency of the precipitation method by using the centrifugal force, which has been used in general as a method of treating a large quantity of cells. However, the centrifugal separation method requires a large scale and expensive apparatus for aseptic treatment and recovery of cells. Moreover, in the centrifugal separation, the types of cells, which can be separated, are limited because the cells have only small differences in their density.
In order to improve the separation potential, there has been employed a density-gradient centrifugation method which uses specific density medium, wherein the specific density of the medium has been strictly adjusted. However, this method cannot simultaneously process a large quantity of cells. In addition, the objective cells must be carefully recovered from the interface which is formed due to the difference in the density. What is worse, the operation for removing unnecessary cell components and the operation for removing unnecessary liquid components must be performed under different conditions. Furthermore, a clean bench must be used to aseptically perform the recovery operation. As described above, the gradient-density centrifugation requires complicated operations.
Moreover, the method (1) may sometimes critically damages the objective cells if an unsuitable centrifugal condition is employed.
The electrical separation method (2) suffers from limited separation efficiency because of the small difference in the static charge on the cell-surface between different cells. Moreover, this method is unsuitable for quickly treating a large quantity of cells. What is worse, this method sometimes may cause damage of objective cells due to application of an electric field on the cells.
Although the affinity separation method (3) has the greatest specificity of the separation methods, an enzymatic process for cleavage of the adsorbed antibody molecules must be performed. Thus, technical problems arise because the enzymatic process damages the cells, the operation cannot easily be performed, and the activity of the antibody cannot easily be maintained. Moreover, the cost is excessively increased because of using expensive antibodies. Thus, this method is unsuitable to quickly treat a large quantity of cells.
The filtration method (4) is a method in which a cell suspension containing the objective cells are allowed to pass through a filter to capture the objective cells on the filter, and then recovery liquid is allowed to pass through the filter in a direction opposite to the foregoing step to detach the captured objective cells from the filter. This method is suitable to quickly separate objective cells in a large quantity from unnecessary cells and liquid components. However, this method has a problem of unsatisfactory efficiency in recovering objective cells. This problem is caused from the fact that the pore size of the filter is constant during the sequential processes for separation.
That is, if the pore size of the filter is enlarged to easily detach the captured objective cells, the quantity of the objective cells which are not captured and allowed to pass through the filter during the filtration is increased. If the pore size of the filter is made to be relatively smaller, the quantity of the objective cells allowed to pass through the filter can be reduced during the filtration and thus the quantity of the captured cells can be increased. However, adhesion properties of the objective cells with respect to the pores is enhanced. As a result, the detachment easiness of the captured cells from the filter is prevented. Thus, high recovery yield cannot be realized, and further, unnecessary cells cannot be removed satisfactorily.
In view of the foregoing problems, filters having appropriate pore size for attaining both requirements i.e., efficiency in capturing objective cells and satisfactory detachment easiness of captured cells, must be selected depending upon purpose of each use.
Incidentally, when the filtration method is performed such that the quantity of pressure of the cell suspension to be supplied for filtration is reduced, the quantity of the objective cells passing through the filter can be reduced to a certain extent and thus the capturing rate can be raised. In this case, however, the quantity which can be processed for unit time is reduced in the foregoing case. Therefore, the advantage of the filtration separation method capable of quickly treating a large quantity cannot be obtained.
On the other hand, when recovery of the objective cells are performed such that the quantity or pressure of the recovery liquid is increased, the detachment or easiness of the captured objective cells from the filter can be improved, resulting in high recovery of the objective cells. In this case, however, the objective cells are damaged considerably and, therefore, the characteristic and the quality of the recovered objective cells deteriorate.
As described above, the conventional methods have merits and demerits with respect to the means for separating the objective cells from unnecessary components and the means for recovering the separated cells. Thus, the conventional methods are used by being selected or combined with each other depending on the purpose and required levels of separation.